Figure 6

Characterization of phosphorylation in platelets downstream of RTL1000. Human washed platelets were exposed to BSA, RTL1000, or fibrinogen (FG) surfaces for 45 min at 37°C. Platelets adherent to RTL1000 or in suspension over BSA were lysed in ice-cold lysis buffer. Platelet lysates were separated by SDS-PAGE and immunoblotted for phosphorylation using A) an anti-phosphotyrosine antibody (4G10), B) anti-Akt antibody, or C) anti-GSK3β antibody. In selected experiments, platelets were pretreated with vehicle or the ADP scavenger apyrase (2 U/ml) in the absence or presence of inhibitors to Src kinases (PP2, 20 μM), PI3 kinases (wortmannin, 100 nM), protein kinase C (R0 31-8220, 20 μM), or Syk kinase (BAY 61-3606, 20 μM).